Bragantia ISSN: 0006-8705 [email protected] Instituto Agronômico de Campinas Brasil

TECHIO, VÂNIA HELENA; CHAMMA DAVIDE, LISETE; CAGLIARI, ALEXANDRO; BARBOSA, SANDRO; VANDER PEREIRA, ANTÔNIO KARYOTIPIC ASYMMETRY OF BOTH WILD AND CULTIVATED OF Bragantia, vol. 69, núm. 2, 2010, pp. 273-279 Instituto Agronômico de Campinas Campinas, Brasil

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KARYOTIPIC ASYMMETRY OF BOTH WILD AND CULTIVATED SPECIES OF PENNISETUM (1)

VÂNIA HELENA TECHIO (2*); LISETE CHAMMA DAVIDE (2); ALEXANDRO CAGLIARI (3); SANDRO BARBOSA (4); ANTÔNIO VANDER PEREIRA (5)

ABSTRACT

This study aimed the establishment of the relation between karyotipic asymmetry values obtained for different accessions of both wild and cultivated species of Pennisetum from Germplasm Bank of Embrapa Gado de Leite/Juiz de Fora-Minas Gerais State, Brazil. Conventional cell cycle synchronization protocols and Feulgen staining method were used to obtain metaphases plates. The wild-type accessions corresponded to the species P. setosum (2n=6x=54), P. nervosum (2n=4x=36), and P. orientale (2n=4x=36), and the cultivated to P. purpureum (2n=4x=28) and P. glaucum (2n=2x=14). No significant difference was found for the total length of chromosomes (p≥0.05) among the species. The analysis of intra-chromosomal asymmetry (A1) and inter-chromosomal asymmetry (A2) has shown that P. setosum has a tendency to chromosome asymmetry. P. nervosum, P. orientale, and P. purpureum have presented an intermediary level of asymmetry and P. glaucum, low asymmetry. Considering Stebbins criteria, the karyotype of P. glaucum and those from the three wild species fitted into the category 1A-symmetrical. With regard to P. purpureum, karyotypes of the accessions BAGs 54, 65 and 91 fitted into the category 2B and the other two genotypes (BAGs 63 and 75) fitted into the 1A. Comparison between the karyotype classification according to the inter- and intra-chromosomal asymmetry and Stebbins methodologies revealed that this last one alone was not able to detect small variations between karyotypes of the taxa closely related. Key words: Pennisetum purpureum, Pennisetum glaucum, germplasm, karyotype.

RESUMO ASSIMETRIA CARIOTÍPICA DE ESPÉCIES SILVESTRES E CULTIVADAS DE PENNISETUM

O objetivo deste estudo foi estabelecer a relação entre os valores de assimetria cariotípica obtidos para diferentes acessos de espécies silvestres e cultivadas de Pennisetum do Banco de Germoplasma da Embrapa Gado de Leite em Juiz de Fora (MG). Para obtenção das metáfases, utilizaram-se os protocolos convencionais de sincronização do ciclo celular e coloração pelo método de Feulgen. Os acessos silvestres correspondem às espécies P. setosum (2n=6x=54), P. nervosum (2n=4x=36) e P. orientale (2n=4x=36) e os cultivados a P. purpureum (2n=4x=28) e P. glaucum (2n=2x=14). Para o comprimento total dos cromossomos não foram observadas diferenças significativas (p≥0,05) entre as espécies. Pela análise da assimetria intra (A1) e intercromossômica (A2) observa-se que P. setosum tem maior tendência à assimetria. P. nervosum, P. orientale e P. purpureum tiveram grau intermediário de assimetria e P. glaucum, menor assimetria. Considerando os critérios de Stebbins, os cariótipos de P. glaucum e das três espécies silvestres analisadas enquadraram-se na categoria 1A-simétrico. Para P. purpureum, os cariótipos dos acessos BAGs 54, 65 e 91 enquadraram-se na categoria 2B e os outros dois genótipos (BAGs 63 e 75), na 1A. A comparação entre as classificações dos cariótipos obtidas conforme as metodologias de assimetria inter e intracromossômica e a de Stebbins revelou que esta última não permitiu detectar pequenas variações entre os cariótipos de táxons proximamente relacionados. Palavras-chave: Pennisetum purpureum, Pennisetum glaucum, germoplasma, cariótipo.

(1) Recebido para publicação em 24 de setembro de 2009 e aceito em 9 de dezembro de 2009. (2) Departamento de Biologia, Universidade Federal de Lavras, 37200-000 Lavras (MG). E-mail: [email protected]; [email protected] (*) Autora correspondente. (3) Departamento de Genética, Universidade Federal do Rio Grande do Sul, 91501-970 Porto Alegre (RS). (4) Departamento de Ciências Biológicas e da Terra, Universidade Federal de Alfenas, 37130-000 Alfenas (MG). (5) Empresa Brasileira de Pesquisa Agropecuária, Embrapa Gado de Leite, 36038-330 Juiz de Fora (MG).

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1. INTRODUCTION elephant grass (P. purpureum) (BURTON, 1944); with P. orientale (PATIL and SINGH, 1964; DUJARDIN and HANNA, The development of actions that promote a 1983b), with P. setaceum (HANNA, 1979), P. dubium better conservation and use of genetic resources has (GILDENHUYS and BRIX, 1958), and with P. squamulatum become a necessity to equilibrate the rational (PATIL et al., 1961; KAUSHAL et al., 2007). DUJARDIN and management of germplasm and to make available the HANNA (1984) also described the formation of tri- material for breeding programs and for biotechnology specific hybrids originated from the breeding between without causing problems of genetic erosion, the millet (P. glaucum), the elephant grass (P. vulnerability and losses of old varieties. In such purpureum) and P. squamulatum. situation, one of the main studied points has been the assessment of genetic diversity and its structure The triploid and hexaploid hybrids between within a certain gene pool. Such question can be better the millet and the elephant grass have been considered understood through studies of domestication as important sources of variation for the selection of processes , the gene flow between different species and superior clones. Characters of forage importance different gene pools and, more particularly, between superior to those found in the parents such as the the wild-type and cultivated forms. These studies may presence of larger leaves in greater number, more involve the analysis of parameters associated with developed stem, smoother leaf hairs and less fibrous recombination, forms of reproduction, adaptation and stems and greater level of dry matter are observed in even the cytogenetic traits (KHALFALLAH et al., 1993). triploid hybrids (BOGDAN, 1977; PEREIRA, 1998). The analysis of numeric or structural variation The efforts to assess the potential of allele of karyotype in populations of one species – cytotypes transference between species of Pennisetum have been or chromosomal race – or between species may be focused in the apomixis and in the perennial growth useful for determining the differences between taxons habit (DUJARDIN and HANNA, 1983a; DUJARDIN and and infer patterns of divergence between the HANNA, 1989; JAUHAR and HANNA, 1998). However, there populations. Such information is essential for are other desired attributes that may be introduced in clarifying the possible role of chromosomal the genome of cultivated species such as precocity in rearrangements in the evolution of organisms and in vegetative development, the increase of efficiency in the speciation. nutrient use, the fairer distribution of dry matter production during the year, and the increase of fertile Regarding the genus Pennisetum, the species seeds. In this case, knowledge of the cytogenetic traits are classified into five sections – Pennisetum, of wild species of Pennisetum and their relation and Heterostachya, Brevivalvula, Gymnothrix, and Penicillaria similarity with cultivated species may enhance the – (STAPF and HUBBARD, 1934; BRUNKEN, 1977) gathering better use of exotic germplasm in breeding. This study the cultivated and wild ones with population from aimed the establishment of the cytological relation exotic material introduced, adapted to different between different accessions of both wild and edaphic conditions, some apomictic with cultivated species of Pennisetum available in the Active chromosomal basic number of 5, 7, 8, and 9 Germplasm Bank of Embrapa Gado de Leite, Juiz de (SCHMELZER, 1997; JAUHAR and HANNA, 1998). The section Fora, Minas Gerais State, Brazil. Pennisetum gathers the cultivated species from the primary and secondary gene pool, millet (P. glaucum, 2n=2x=14) and elephant grass (P. purpureum, 2. MATERIAL AND METHODS 2n=4x=28), respectively. The chromosomal races or cytotypes P. orientale (2n=18, 27, 36, 45, 54) and P. Fifteen wild accessions and ten cultivated pedicelatum (2n=36, 45, and 54) are found among the accessions (Table 1) of Pennisetum from the Active perennial species of the tertiary gene pool (JAUHAR and Germplasm Bank of Embrapa Gado de Leite, Juiz de HANNA, 1998). Fora, Minas Gerais State, Brazil were assessed. These were sampled in different physiographic areas Despite the existence of mechanisms that of Brazil and identified as BAG (Active Germplasm contribute for the maintenance of this genetic Bank) followed by the number of its record. structure, such as linkage and gametophytic competition (ROBERT et al., 1991), some species are not The cytogenetic analyses were carried in sexually compatible. This possibility of interspecific meristematic cells obtained from root edges. The combination is important for breeding as it allows the material was submitted to cell cycle synchronization transference of alleles into species of agronomical using a 2.5 mM hydroxyurea solution for 14h. The importance. Previous authors have described the roots were pretreated with cycleheximid solution (25 formation of the inter-specific hybrids between the mg/L) and 8-hidroxyquinoline (300 mg/L) (1:1) for millet and other species of Pennisetum like with the 2h45min, fixed in Carnoy solution for 24h, and

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submitted to enzymatic maceration with pectinase by STEBBINS (1971) and ZARCO (1986) were used for the (SIGMATM) diluted in citrate buffer, pH 4.6 (5:1) for karyotypic symmetry. 2h45min (TECHIO et al., 2002). The staining was carried The analysis of variance followed by the test of through the Feulgen method and the slides prepared SCOTT KNOTT (1974), employing the level of significance according to the smear technique. The material p>0.05, were used for comparison of the variation herborized is conserved at the Herbarium ESAL of the magnitude regarding to the chromatin total length (CTC). Biology Department of the Universidade Federal de Lavras (UFLA), Lavras, Minas Gerais State, Brazil. The chromosomal measures were performed 3. RESULTS AND DISCUSSION using the software Image Tool (WILCOX et al., 2002). The plants analysed corresponded to the The chromosomal data of the accessions of P. glaucum species P. setosum (accessions 3, 4, 5, and 6), with and P. purpureum were obtained from BARBOSA et al. 2n=6x=54; P. nervosum (accessions 1, 7, 8, 9, 10, 12, (2003). The centromeric index (IC=[BC/(BC+BL)]x100) 13, 14, 16, and 17), with 2n=4x=36, and P. orientale was calculated for all accessions and the chromosomes (accession 15), with 2n=4x=36 chromosomes (Table 1 were classified according to the centromere position and Figure 1). BARBOSA et al. (2003) have confirmed in metacentric – m (IC between 50 and 37.5) – and for P. purpureum (BAG 54, 63, 65, 75, and 91) and for submetacentric – sm (IC between 37.5 and 25) – P. glaucum (BAG M 24, 35, 36, 38, and 44), 2n=4x=28 according to LEVAN et al. (1964). The criteria proposed and 2n=2x=14 chromosomes, respectively (Figure 1).

Table 1. Pennisetum acessions of Active Germplasm Bank of Embrapa Gado de Leite. Juiz de Fora, Minas Gerais State, Brazil.

Accession Scientific name Common name Place BAG Selvagem 3 P. setosum - Distrito Federal BAG Selvagem 4 P. setosum - Mato Grosso BAG Selvagem 5 P. setosum - Mato Grosso BAG Selvagem 6 P. setosum - Mato Grosso BAG Selvagem 1 P. nervosum - Mato Grosso do Sul BAG Selvagem 7 P. nervosum - Mato Grosso do Sul BAG Selvagem 8 P. nervosum - Mato Grosso do Sul BAG Selvagem 9 P. nervosum Bufão Goiás BAG Selvagem 10 P. nervosum - Goiás BAG Selvagem 12 P. nervosum - Unavailable BAG Selvagem 13 P. nervosum - Mato Grosso do Sul BAG Selvagem 14 P. nervosum - Rio Grande do Sul BAG Selvagem 16 P. nervosum - Mato Grosso do Sul BAG Selvagem 17 P. nervosum - Mato Grosso do Sul BAG Selvagem 15 P. orientale - Mato Grosso do Sul BAG 54 P. purpureum Capim cana d’África Unavailable BAG 63 P. purpureum Cuba 169 Unavailable BAG 65 P. purpureum Roxo Botucatu Unavailable BAG 75 P. purpureum IJ 7136 Unavailable BAG 91 P. purpureum - Unavailable M24 P. glaucum ICMB 90111 Unavailable M35 P. glaucum - Unavailable M36 P. glaucum AFPOP88 Unavailable M38 P. glaucum AFPOP 90 Unavailable M44 P. glaucum - Unavailable

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The accessions of P. nervosum have shown a mean length of 1.17 µm for the chromosome 1, which represents in average 3% of the complement, and for the shortest chromosome such values were 0.90 µm and 2.3%. In P. orientale, the length of the longest chromosome (1.09 µm) represents 3.7% of the complement and the shortest (0.54 µm), 1.8%. In the hexeploid accessions of P. setosum, the longest (1.15 µm) and the shortest (0.70 µm) have represented 2.3% and 1.4% of the complement, respectively. For the accessions BAG 54, 65, and 91 of P. purpureum, BARBOSA et al. (2003) have described that the longest chromosomal pair represents, in average, 10.7% of the length of the haploid lot, being twice as long as the last pair, whose relative length represents, in average, 5% of the haploid lot. Among the accessions Figure 1. Mitotic metaphases A. Pennisetum nervosum BAG 63 and 75, the relation between the longest and (2n=4x=36); B. Pennisetum setosum (2n=6x=54); C. shortest chromosome has not reached the proportion Pennisetum orientale (2n=4x=36); D. Pennisetum 2:1. In P. glaucum, the authors have described that purpureum (2n=4x=28); E. Pennisetum glaucum the longest pair represented, in average, 17.1% of the (2n=2x=14). Bars correspond to 5 µm. haploid lot, being 1.6 times longer than the last pair, whose relative length represented, in average, 10.9% of the haploid set. The analysis of variance followed by the test The karyotypes of P. orientale and P. glaucum of Scott Knott confirmed the absence of significant showed metacentric and submetacentric chromosomes, difference (p≥0,05) for the total length of chromosomes while the karyotype of P. setosum, P. nervosum, and P. between the wild accessions of P. orientale (29.41 purpureum have shown only metacentric chromosomes µm±2.82), P. nervosum (38.72 µm±4.39) and P. setosum (Table 2). The high level of chromosomal (49.43 µm±4.18) and the cultivated species P. condensation and the similarities in chromosomal purpureum (28.09 µm±1.69) and P. glaucum (28.36 sizes hindered the identification of morphologic traits, µm±3.92). besides the centromere, being only possible the visualization of satellites in one pair of chromosomes Considering that in a nucleus the total mass in the accessions of P. nervosum. Those numbers are, of chromosomes is strictly correlated with the DNA probably, underestimated, since due to their terminal content, STEBBINS (1971) reported the possibility of or interstitial positioning, the secondary constrictions inferences upon the DNA content of an organism are not easily detectable through conventional staining through the chromosomal analysis. Based on this techniques. In the accessions of P. glaucum and P. criterion, no substantial difference was found in the purpureum, BARBOSA et al. (2003) described the presence amount of DNA among those three wild species, of satellites in one or two pairs of chromosomes. whose basic chromosomal number is x=9, and cultivated species with x=7. Results obtained for the Using the criteria proposed by ZARCO (1986), total chromosomal length of wild species coincided the data analysis from chromosomal measurements with the data of DNA quantification obtained through suggest that karyotypes of the accessions/species, flow cytometry by MARTEL et al. (1997), who found even considerably similar, may be distinguished with that species of Pennisetum with x=9 showed similar regard to chromosomal length and karyotypic amount of DNA per haploid genome, varying from 0.85 asymmetry. The analysis of the intra-chromosomal to 0.95pg, independent of the level of ploidy. The asymmetry (A1) and the inter-chromosomal cultivated species of P. glaucum and P. purpureum, both asymmetry (A2) have shown that P. setosum (x=6) with x=7, have 2.36 and 1.15 pg per haploid set presents more tendency to asymmetry, with the respectively. In the triploid hybrid (P. glaucum x P. following mean indexes A1=0.968 and A2=0.161. The purpureum), CAMPOS et al. (2009) observed that the tetraploid accessions of P. nervosum, P. orientale, and amount of DNA per genome was 1.50 pg. In P. purpureum have exhibited intermediary level of Pennisetum, cytologic and flow cytometry studies have asymmetry and the karyotype of P. glaucum (x=7) evidenced that the size of genome and chromosomes presented the smallest asymmetry, represented by are negatively related to the chromosomal basic mean values of A1=0.898 and A2=0.162 (Table 1, number (JAHUAR, 1981; MARTEL et al., 1997). Figure 1).

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Table 2. Karyotypic traits of accessions of Pennisetum nervosum, P. setosum, and P. orientale, P. purpureum and P. glaucum: chromosomal number, karyotypic formulae, total length of the chromatin (CTC), chromosomal mean length (CMC); length of the longest (L) and shortest (S) chromosomes of each species; mean centromeric index (IC) and mean indexes of karyotypic asymmetry (A1) and inter-chromosomal asymmetry (A2), according to ZARCO (1986)

Chromosomal Karyotypic CTC Species CMC L S IC A1 A2 number formulae 2n x µm P. setosum 2n=6x=54 54 m 49.43 8.24 0.91 1.15 0.70 45.80 0.968 0.161 P. nervosum 2n=4x=36 36 m 38.72 9.68 1.07 1.17 0.90 46.68 0.953 0.164 P. orientale 2n=4x=36 32 m + 4 sm 29.41 7.36 0.82 1.09 0.54 43.16 0.954 0.145 P.purpureum 2n=4x=28 28 m (1) 28.09 7.02 2.001 2.941 1.471 44.891 0.941 0.194 P. glaucum 2n=2x=14 10 m + 4 sm (1) 28.36 14.18 4.021 4.861 3.061 40.741 0.898 0.162

1 ( ) BARBOSA et al. (2003).

Considering the criteria proposed by STEBBINS genus belong to the group that presents a (1971) for the definition of symmetry, the karyotypes chromosomal basic number equal to nine (x=9), of the three wild species analysed fit into the category small chromosomes, apomictic mode of 1A-symmetrical, since the relation between the longest reproduction and perennial life cycle. The species and shortest was lower than 2:1. Regarding P. with x= 5, 7 and 8 would be those that diverged purpureum, BARBOSA et al. (2003) suggested the more recently, indicating that the structure of the inclusion of the karyotype of the accessions BAG 54, genome of Pennisetum must be involved in the 65, and 91 in the category 2B and accessions BAG 63 reduction of chromosome number and increase of and 75, in the category 1A. The karyotypes of the their size (MARTEL et al., 2004). accessions of P. glaucum were included in the Observing the spatial arrangement between category 1A. the analysed populations in the Figure 2, it is verified The proposal of STEBBINS (1971), supported by that polyploid species (P. setosum, P. nervosum, P. the concepts of the Russian School lead by LEVITSKY orientale, and P. purpureum) are limited to closer (1931), define that symmetry of the karyotype is areas in the graphic of dispersions, indicating more characterized by the predominance of chromosomes karyotypic homogeneity between them and a better similar in length and with centromere in metacentric possibility of gene exchange, given that there is and submetracentric positions. The increase of sexual compatibility. Studies regarding gene karyotypic asymmetry occurs due to the change of the structure show that the distribution of genetic centromere to terminal or subterminal positions, and variability is not random within the populations to differences in the relative size of the chromosomes and may be determined by characteristics such as of the complement, making the karyotype more geographic distribution of the species, gene flow, heterogeneous. reproductive system, effective size of the The comparison between the classifications of populations and more, a set of evolutive factors, karyotypes obtained according to the methodology of such as migration, mutation, natural selection and ZARCO (1986) and STEBBINS (1971) revealed that this gene derivation (WEIR, 1996). In other forage species, last one does not allow the detection of little variations Lolium perene (SWEENEY and DANNENBERGER, 1994), among the karyotype closely related, making the Lolium multiflorum (VIEIRA et al., 2004), and Trifolium classification inaccurate. Such finding ratified the pratense (KONGKIATNGAM et al., 1995), a higher intra- critics stated by ZARCO (1986) and PASZKO (2006) population variability rather than an regarding to the amplitude of categories proposed by inter-population variability was detected. STEBBINS (1971). Considering the closer karyotypic proximity Generally, the species with more symmetric between P. purpureum and the other polyploid species, karyotypes and higher number of chromosomes are the promotion of breeding between them and the considered more primitive in a certain group of assessment of performance, agronomic behaviour, and . For Pennisetum, however, it has been widely level of fertility of these hybrids would be reported (AVDULOV, 1931; PANTULU and RAO, 1982; recommended in comparison to those obtained from MARTEL et al., 2004;) that the ancestral traits of the the breeding with P. glaucum.

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BOGDAN, A. V. Tropical pasture and fodder plants (grasses and legumes). London: Longman, 1977. 241p.

BRUNKEN, J. N. A systematic study of Pennisetum Sect. Pennisetum (Gramineae). American Journal of Botany, v.64, p.161-176, 1977.

BURTON, G. W. Hybrids between napier grass and cattail millet. Journal of Heredity, v.35, p.227-232, 1944.

CAMPOS, J. M. S.; CALDERANO, C.A.; PEREIRA, A.V.; DAVIDE, L. C.; VICCINI, L. F.; SANTOS, M. O. Embriogênese somática em híbridos de Pennisetum sp. e avaliação de estabilidade genômica por citometria. Pesquisa Agropecuária Brasileira, v.44, p.38-44, 2009.

DUJARDIN, M.; HANNA, W. W. Apomictic and sexual pearl Figure 2. Karyotypic asymmetry according to ZARCO millet x Pennisetum squamulatum hybrids. The Journal of (1986). Accessions of P. glaucum - 2n=2x=14 (¢); P. Heredity, v. 74, p. 277-279, 1983a. purpureum – 2n=4x=28 (p); P. nervosum – 2n=4x=36 (¿); P. orientale - 2n=4x=36 (*) and P. setosum - 2n=6x=54 DUJARDIN, M.; HANNA, W. W. Meiotic and reproductive (˜). A1= intra-chromosomal asymmetry and A2= inter- behavior of facultative apomictic BC1 offspring derived from chromosomal asymmetry. Pennisetum americanum-P. orientale interspecific hybrids. Crop Science, v.23, p.156-160, 1983b.

4. CONCLUSIONS DUJARDIN, M.; HANNA, W. W. Cytogenetics of double cross hybrids between Pennisetum americanum-P. purpureum 1. No difference was found for the total length amphiploids and P. americanum x P. squamulatum interspecific of the chromosomes between the wild species (x=9) hybrids. Theoretical and Applied Genetics, v.69, p.97-100, 1984. and the cultivated species (x=7). DUJARDIN, M.; HANNA, W. W. Crossability of pearl millet 2. The tetraploid accessions of P. setorum, P. with Pennisetum species. Crop Science, v.29, p.77-80, 1989. nervosum, P. orientale and P. purpureum have GILDENHUYS, P.; BRIX, K. Cytological abnormalities in presented an intermediary level of asymmetry and Pennisetum dubium. Heredity, v.12, p.441-452, 1958. higher karyotypic homogeneity and the karyotype of P. glaucum (x=7), a lower asymmetry. HANNA, W. W. Interspecific hybrids between pearl millet and fountain grass. Journal of Heredity, v.70, p.425-427, 1979. 3. The indexes of both inter- and intra- chromosomal asymmetry allow the detection of low JAUHAR, P. P. Cytogenetics and breeding of pearl millet and variations between the karyotypes of the taxa closely related species. New York: Liss A. R., 1981. 289p. related, helping in the identification of the accessions JAUHAR, P. P.; HANNA, W. W. Cytogenetics and genetics of of Pennisetum sp. in the germplasm banks. pearl millet. Advances in Agronomy, v.64, p.1-26, 1998.

KAUSHAL, P.; ROY, A. K.; KHARE, A.; MALAVIYA, D. R.; ACKNOWLEDGMENT ZADOO, S. N.; CHOUBEY, R. N. Crossability and characterization of interspecific hybrids between sexual Authors gratefully acknowledge the Fundação Pennisetum glaucum (pearl millet) and a new cytotype (2n=56) de Amparo à Pesquisa do Estado de Minas Gerais for of apomictic P. Squamulatum. Cytologia, v.72, p.111-118, 2007. the financial support, and the biologist Larissa Fonseca Andrade Vieira by contributions in obtaining KHALFALLAH, N.; SARR, A.; SILJAK-YAKOVLEV, S. Karyological study of some cultivated and wild stocks of pearl the photomicrographs. millet from Africa (Pennisetum typhoides Stapf et Hubb. And P. violaceum (Lam.) L. Rich. Caryologia, v.46, p.127-138, 1993.

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